Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering

Ammonia is a ubiquitous waste product of protein metabolism that can accumulate in numerous metabolic disorders, causing neurological dysfunction ranging from cognitive impairment to tremor, ataxia, seizures, coma and death. The brain is especially vulnerable to ammonia as it readily crosses the blood-brain barrier in its gaseous form, NH3, and rapidly saturates its principal removal pathway located in astrocytes. Thus, we wanted to determine how astrocytes contribute to the initial deterioration of neurological functions characteristic of hyperammonemia in vivo. Using a combination of two-photon imaging and electrophysiology in awake head-restrained mice, we show that ammonia rapidly compromises astrocyte potassium buffering, increasing extracellular potassium concentration and overactivating the Na+-K+-2Cl− cotransporter isoform 1 (NKCC1) in neurons. The consequent depolarization of the neuronal GABA reversal potential (EGABA) selectively impairs cortical inhibitory networks. Genetic deletion of NKCC1 or inhibition of it with the clinically used diuretic bumetanide potently suppresses ammonia-induced neurological dysfunction. We did not observe astrocyte swelling or brain edema in the acute phase, calling into question current concepts regarding the neurotoxic effects of ammonia. Instead, our findings identify failure of potassium buffering in astrocytes as a crucial mechanism in ammonia neurotoxicity and demonstrate the therapeutic potential of blocking this pathway by inhibiting NKCC1.

[1]  N. Matsuki,et al.  GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy , 2012, Nature Medicine.

[2]  C. Rose,et al.  Kir4.1 channels mediate a depolarization of hippocampal astrocytes under hyperammonemic conditions in situ , 2012, Glia.

[3]  Michael Chen,et al.  Forebrain engraftment by human glial progenitor cells enhances synaptic plasticity and learning in adult mice. , 2013, Cell stem cell.

[4]  R. Butterworth,et al.  Effects of congenital hyperammonemia on the cerebral and hepatic levels of the intermediates of energy metabolism in spf mice. , 1992, Biochemical and biophysical research communications.

[5]  S. Wall,et al.  NH+4 transport mediated by Na(+)-K(+)-ATPase in rat inner medullary collecting duct. , 1994, The American journal of physiology.

[6]  E. Delpire,et al.  Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter , 1999, Nature Genetics.

[7]  J. Marrero,et al.  Azoxymethane-induced fulminant hepatic failure in C57BL/6J mice: characterization of a new animal model. , 1999, The American journal of physiology.

[8]  M. Norenberg,et al.  Na-K-Cl Cotransporter-1 in the Mechanism of Ammonia-induced Astrocyte Swelling* , 2008, Journal of Biological Chemistry.

[9]  M. Nedergaard,et al.  ‘Hit & Run’ Model of Closed-Skull Traumatic Brain Injury (TBI) Reveals Complex Patterns of Post-Traumatic AQP4 Dysregulation , 2013, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[10]  P. Marcaggi,et al.  Neuron–glial trafficking of NH4+ and K+: separate routes of uptake into glial cells of bee retina , 2004, The European journal of neuroscience.

[11]  P. Frankland,et al.  Computer-assisted behavioral assessment of Pavlovian fear conditioning in mice. , 2000, Learning & memory.

[12]  W. Raabe Ammonia and disinhibition in cat motor cortex by ammonium acetate, monofluoroacetate and insulin-induced hypoglycemia , 1981, Brain Research.

[13]  R. Butterworth,et al.  Effect of ammonium ions on synaptic transmission in the mammalian central nervous system , 1992, Progress in Neurobiology.

[14]  D. Tank,et al.  Imaging Large-Scale Neural Activity with Cellular Resolution in Awake, Mobile Mice , 2007, Neuron.

[15]  Robert C. Wolpert,et al.  A Review of the , 1985 .

[16]  T. Doetschman,et al.  Mice Lacking the Basolateral Na-K-2Cl Cotransporter Have Impaired Epithelial Chloride Secretion and Are Profoundly Deaf* , 1999, The Journal of Biological Chemistry.

[17]  C. Xiao,et al.  Taurine activates excitatory non‐synaptic glycine receptors on dopamine neurones in ventral tegmental area of young rats , 2005, The Journal of physiology.

[18]  T. Takano,et al.  Astrocytes Modulate Neural Network Activity by Ca2+-Dependent Uptake of Extracellular K+ , 2012, Science Signaling.

[19]  Arne Klungland,et al.  Glial-conditional deletion of aquaporin-4 (Aqp4) reduces blood–brain water uptake and confers barrier function on perivascular astrocyte endfeet , 2011, Proceedings of the National Academy of Sciences.

[20]  H. Lux Ammonium and Chloride Extrusion: Hyperpolarizing Synaptic Inhibition in Spinal Motoneurons , 1971, Science.

[21]  K. Sato,et al.  The differential expression patterns of messenger RNAs encoding K-Cl cotransporters (KCC1,2) and Na-K-2Cl cotransporter (NKCC1) in the rat nervous system , 2001, Neuroscience.

[22]  R. Butterworth Pathophysiology of Hepatic Encephalopathy: A New Look at Ammonia , 2002, Metabolic Brain Disease.

[23]  J. A. Payne,et al.  The K+/Cl− co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation , 1999, Nature.

[24]  Yoshiaki Yamamoto,et al.  Risk factors for hyperammonemia in pediatric patients with epilepsy , 2013, Epilepsia.

[25]  J. Poulet,et al.  Facilitating sensory responses in developing mouse somatosensory barrel cortex. , 2007, Journal of neurophysiology.

[26]  R. A. Waniewski,et al.  Physiological Levels of Ammonia Regulate Glutamine Synthesis from Extracellular Glutamate in Astrocyte Cultures , 1992, Journal of neurochemistry.

[27]  B. Mallick,et al.  Comparison of Na-K ATPase activity in rat brain synaptosome under various conditions. , 1998, Neurochemistry International.

[28]  M. Norenberg,et al.  Glutamine synthetase: glial localization in brain , 1977, Science.

[29]  D. Leibfritz,et al.  Selective increase of brain lactate synthesis in experimental acute liver failure: Results of a [1H‐13C] nuclear magnetic resonance study , 2003, Hepatology.

[30]  R. Miles,et al.  Perturbed Chloride Homeostasis and GABAergic Signaling in Human Temporal Lobe Epilepsy , 2007, The Journal of Neuroscience.

[31]  P. Hannaert,et al.  Rat NKCC2/NKCC1 cotransporter selectivity for loop diuretic drugs , 2002, Naunyn-Schmiedeberg's Archives of Pharmacology.

[32]  E. Hoffman,et al.  Gene expression profiling of astrocytes from hyperammonemic mice reveals altered pathways for water and potassium homeostasis in vivo , 2008, Glia.

[33]  R. Butterworth,et al.  Na+,K+-ATPase activities are increased in brain in both congenital and acquired hyperammonemic syndromes , 1995, Neuroscience Letters.

[34]  Steven Goldman,et al.  Gap-junction-mediated propagation and amplification of cell injury , 1998, Nature Neuroscience.

[35]  K. Okamoto,et al.  EFFECTS OF AMMONIUM IONS ON SPONTANEOUS ACTION POTENTIALS AND ON CONTENTS OF SODIUM, POTASSIUM, AMMONIUM AND CHLORIDE IONS IN BRAIN IN VITRO , 1978, Journal of neurochemistry.

[36]  J. Seegmiller,et al.  An enzymatic determination of ammonia in biological fluids. , 1965, The Journal of laboratory and clinical medicine.

[37]  F. Jensen,et al.  NKCC1 transporter facilitates seizures in the developing brain , 2005, Nature Medicine.

[38]  W. Raabe,et al.  Disinhibition in cat motor cortex by ammonia. , 1975, Journal of neurophysiology.

[39]  O. Braissant,et al.  Hyperammonemia-induced toxicity for the developing central nervous system , 2007, Brain Research Reviews.

[40]  J. L. Stringer,et al.  Sodium pump activity, not glial spatial buffering, clears potassium after epileptiform activity induced in the dentate gyrus. , 2000, Journal of neurophysiology.

[41]  Arthur J. L. Cooper 13N as a tracer for studying glutamate metabolism , 2011, Neurochemistry International.

[42]  B. Mallick,et al.  Rapid communication Comparison of Na-K ATPase activity in rat brain synaptosome under various conditions , 1998, Neurochemistry International.

[43]  J. Rothstein,et al.  Variations in Promoter Activity Reveal a Differential Expression and Physiology of Glutamate Transporters by Glia in the Developing and Mature CNS , 2007, The Journal of Neuroscience.

[44]  Alcino J. Silva,et al.  Memory for context becomes less specific with time. , 2007, Learning & memory.

[45]  Charles Nicholson,et al.  Ion-selective microelectrodes and diffusion measurements as tools to explore the brain cell microenvironment , 1993, Journal of Neuroscience Methods.

[46]  Ø. Skare,et al.  Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema , 2010, Proceedings of the National Academy of Sciences.

[47]  S. Javaheri,et al.  Ionic Composition of Cisternal CSF in Acute Respiratory Acidosis: Lack of Effect of Large Dose Bumetanide , 1993, Journal of neurochemistry.

[48]  M. Nedergaard,et al.  Real-time analysis of microglial activation and motility in hepatic and hyperammonemic encephalopathy , 2012, Neuroscience.

[49]  K. Ballanyi,et al.  Disruption of KCC2 Reveals an Essential Role of K-Cl Cotransport Already in Early Synaptic Inhibition , 2001, Neuron.

[50]  Fahmeed Hyder,et al.  Focal BOLD fMRI changes in bicuculline-induced tonic–clonic seizures in the rat , 2010, NeuroImage.

[51]  G. Garden,et al.  A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia , 2010, Journal of visualized experiments : JoVE.

[52]  M. Robinson,et al.  Adenovirus-Mediated in Vivo Gene Transfer Rapidly Protects Ornithine Transcarbamylase-Deficient Mice from an Ammonium Challenge , 1997, Pediatric Research.

[53]  S. Chew,et al.  Ammonia Production, Excretion, Toxicity, and Defense in Fish: A Review , 2010, Front. Physiology.

[54]  George J Augustine,et al.  Progressive NKCC1-Dependent Neuronal Chloride Accumulation during Neonatal Seizures , 2010, The Journal of Neuroscience.

[55]  T. Takano,et al.  Cortical spreading depression causes and coincides with tissue hypoxia , 2007, Nature Neuroscience.

[56]  Nanhong Lou,et al.  General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex , 2012, Proceedings of the National Academy of Sciences.